Establishing a new blood supply, known as neovascularization, is important for tumor growth and metastasis (Folkman 1990). Formation of blood vessels is a complex process involving endothelial cell proliferation, matrix degradation, migration, tube formation and maturation. Growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) induce endothelial cell proliferation and migration (Ferrara et al. 1992; Kandel et al. 1991). In addition to growth factor receptor-mediated signaling, interaction between cell surface anchored integrins and extracellular matrix components constitute an additional pathway necessary for angiogenesis. In fact, recent studies have identified two cytokine mediated, integrin dependent angiogenic pathways. One of these pathways is associated with xcex1vxcex23 integrin, which selectively influences basic FGF (bFGF) mediated angiogenic signals (Friedlander et al. 1995). A second non-overlapping pathway is represented by crosstalk between xcex1vxcex25 integrin and protein kinase C (PKC) dependent growth factor mediated signaling (VEGF, bFGF, IGF, TNF-xcex1) (Friedlander et al. 1995; Brooks et al. 1997). Tumor angiogenesis can therefore be inhibited either by blocking the interaction between xcex1vxcex23/xcex25 and arginine-glycine-aspartate (RGD) containing extracellular matrix or by interfering with angiogenic growth factors. A number of these strategies are currently under clinical development.
A number of proteolytic fragments of endogenous proteins and factors are capable of inhibiting angiogenesis (Folman 1995; Dawson et al. 1999). The former group of inhibitors includes angiostatin, encompassing kringle 1-4 of plasminogen, endostatin, a proteolytic fragment of a collagen type XVIII, and a fragment of antithrombin (Folman 1995; O""Reilly et al. 1997; O""Reilly et al. 1999). The molecular mechanism of action of angiostatic (antiangiogenic) proteins is not completely understood. Endostatin, for example, inhibits endothelial cell (EC) migration and induces apoptosis by modulating the antiapoptotic protein, BCL-2 (Dhanabal et al. 1999). A direct interaction with matrix metalloproteinases has also been suggested to be responsible for the antiangiogenic activity of endostatin (Kim et al. 2000). Endostatin treatment has been shown to inhibit solid tumor growth in a number of model systems (O""Reilly et al. 1997; Dhanabal et al. 1999; Boehm et al. 1997). O""Reilly et al. (1997) also reported that endostatin treatment resulted in complete regression of established tumors in some instances. However, the in vivo use of antiangiogenic proteins requires daily administration for prolonged periods of time. Improving the potency of these inhibitors would therefore significantly enhance the clinical use of these novel therapeutic molecules.
Therefore, what is needed are improved antiangiogenic proteins, i.e., proteins with increased potency so that higher efficacy may be achieved by lower doses.
The present invention provides a composition having a targeting moiety specific for endothelial cells linked to an antiangiogenic moiety. A xe2x80x9ctargeting moietyxe2x80x9d as used herein is a molecule that facilitates the interaction and/or binding of the linked antiangiogenic moiety to endothelial cells. Preferably, the targeting moiety and the antiangiogenic moiety are polypeptides or peptides, which, when linked form a chimeric polypeptide. The term xe2x80x9cchimeric polypeptidexe2x80x9d refers to a protein that includes amino acid sequences or segments that are positioned or linked in a manner which does not normally occur in the native genome of a species. More preferably, the targeting moiety is a polypeptide or peptide derived from a particular extracellular matrix (ECM) polypeptide, such as human fibronectin. Fibronectin contains the tripeptide RGD (Arg-Gly-Asp), which is also found within most major types of matrix proteins. A xe2x80x9cpeptidexe2x80x9d as used herein has fewer than 20 residues, e.g., fewer than 10 or fewer than 5 residues. Preferred targeting moieties bind to xcex1vxcex23/xcex1vxcex25 integrins, e.g., RGD is a motif found in molecules that bind xcex1vxcex23, and/or to xcex15 and/or xcex1v integrins, as endostatin may interact with xcex15 and/or xcex1v integrins as well (Rehn et al., 2001). Thus, preferred peptide or polypeptide targeting moieties are derived from RGD-containing molecules such as vitronectin, osteopontin bone sialoprotein, and disintegrins, as well as other molecules which are specific for xcex1vxcex23, e.g., echistatin, kistrin, integrelin, tirofiban, amifiban or xemolofiban, including anti-xcex1vxcex23 antibodies. Preferred targeting moieties include but are not limited to RGD, NGR, RGDNGR (SEQ ID NO:8), NGRRGD (SEQ ID NO:9), or tandom repeats of RGD, NGR, RGDNGR (SEQ ID NO:8), or NGRRGD (SEQ ID NO:9), or any combination thereof. Preferred polypeptide or peptide targeting moieties are less than about 100, more preferably less than about 50, even more preferably less than about 10, but at least 3, residues in length, and when linked to the antiangiogenic polypeptide yield a chimeric polypeptide that inhibits proliferation and/or migration of endothelial cells, attaches to endothelial cells, and/or inhibits tumor growth, and preferably is enhanced in these properties relative to the corresponding non-chimeric antiangiogenic polypeptide. In one embodiment of the invention, the targeting moiety is RGD.
The targeting moiety can be linked to the amino or the carboxyl terminus, or both, of the antiangiogenic polypeptide, e.g., by covalent bonding, and a different targeting moiety can be present at the amino terminus than at the carboxyl terminus. Preferred anti-angiogenic polypeptides are endostatin and angiostatin. For example, a chimeric endostatin, e.g., human endostatin genetically modified to include an RGD-motif at the amino or carboxyl terminus, is provided. As described hereinbelow, in vitro cell-binding studies showed that endothelial cells had enhanced binding to RGD-endostatin coated plates relative to native (unmodified) endostatin. The enhanced binding was completely blocked by anti xcex1vxcex23 antibody or RGD peptide. Endostatin-RGD was more potent in inhibiting bFGF-induced endothelial cell proliferation when compared to native endostatin. Moreover, RGD-modified endostatins were more potent in inhibiting endothelial cell migration when compared to native endostatin. RGD-containing endostatins were also more effective in inhibiting tumor growth in athymic nude mice. Further, a slow (sustained) release formulation comprising alginate beads and endostatin was very effective even at a fraction of the dose given in bolus injections. In particular, alginate beads containing the modified endostatin completely inhibited established ovarian cancers in athymic mice. Thus, the antiangiogenic activity of endostatin can be improved by adding an RGD-sequence.
The invention further provides a composition comprising a targeting moiety specific for endothelial cells linked to an antiangiogenic moiety and a pharmaceutically acceptable diluent. Also provided is a sustained release dosage form comprising a composition of the invention.
The invention also provides a recombinant polynucleotide encoding a chimeric polypeptide of the invention, host cells transformed with such a recombinant polynucleotide and the use of the composition, recombinant polynucleotide, e.g., in a recombinant virus, or transformed host cell of the invention, e.g., to inhibit or prevent undesirable endothelial cell proliferation and/or migration.
Further provided are methods to inhibit or prevent undesirable endothelial cell proliferation and/or migration. For example, a method of the invention involves contacting a mammalian endothelial cell with an amount of a composition of the invention effective to inhibit or prevent undesirable endothelial cell proliferation. Mammalian cells include primate cells such as human cells, rodent cells, e.g., mouse, hamster or rat, bovine cells, canine cells, feline cells, swine cells, equine cells, caprine cells, ovine cells and the like.
The present invention further provides therapeutic methods, which involve administering to a mammal having, or at risk of, a condition characterized by undesirable endothelial cell proliferation, an effective amount of a composition or a sustained release dosage form of the invention. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. For example, a condition characterized by undesirable endothelial cell proliferation can be cancer, e.g., colon cancer, ovarian cancer, breast cancer or hematologic malignancies, diabetic retinopathy, rheumatoid arthritis, psoriasis, macular degeneration, restenosis, or eye disorders related to endothelial cell proliferation.